SK Channels Research Articles

Gating kinetics and pharmacological properties of small-conductance Ca2+-activated potassium channels

Small-conductance (SK) calcium-activated potassium channels are a promising treatment target in atrial fibrillation. However, the functional properties that differentiate SK inhibitors remain poorly understood. The objective of this study was to determine how two unrelated SK channel inhibitors, apamin and AP14145, impact SK channel function in excised inside-out single-channel recordings. Surprisingly, both apamin and AP14145 exert much of their inhibition by inducing a class of very-long-lived channel closures (apamin: τc,vl = 11.8 ± 7.1 s, and AP14145: τc,vl = 10.3 ± 7.2 s), which were never observed under control conditions. Both inhibitors also induced changes to the three closed and two open durations typical of normal SK channel gating. AP14145 shifted the open duration distribution to favor longer open durations, whereas apamin did not alter open-state kinetics. AP14145 also prolonged the two shortest channel closed durations (AP14145: τc,s = 3.50 ± 0.81 ms, and τc,i = 32.0 ± 6.76 ms versus control: τc,s = 1.59 ± 0.19 ms, and τc,i = 13.5 ± 1.17 ms), thus slowing overall gating kinetics within bursts of channel activity. In contrast, apamin accelerated intraburst gating kinetics by shortening the two shortest closed durations (τc,s = 0.75 ± 0.10 ms and τc,i = 5.08 ± 0.49 ms) and inducing periods of flickery activity. Finally, AP14145 introduced a unique form of inhibition by decreasing unitary current amplitude. SK channels exhibited two clearly distinguishable amplitudes (control: Ahigh = 0.76 ± 0.03 pA, and Alow = 0.54 ± 0.03 pA). AP14145 both reduced the fraction of patches exhibiting the higher amplitude (AP14145: 4/9 patches versus control: 16/16 patches) and reduced the mean low amplitude (0.38 ± 0.03 pA). Here, we have demonstrated that both inhibitors introduce very long channel closures but that each also exhibits unique effects on other components of SK gating kinetics and unitary current. The combination of these effects is likely to be critical for understanding the functional differences of each inhibitor in the context of cyclical Ca2+-dependent channel activation in vivo.

SK and Kv4 Channels Limit Spike Timing Perturbations in Pacemaking Dopamine Neurons.

Midbrain dopamine (DA) neurons are among the best characterized pacemaker neurons, having intrinsic, rhythmic firing activity even in the absence of synaptic input. However, the mechanisms of DA neuron pacemaking have not been systematically related to how these cells respond to synaptic input. The input-output properties of pacemaking neurons can be characterized by the phase-resetting curve (PRC), which describes the sensitivity of interspike interval (ISI) length to inputs arriving at different phases of the firing cycle. Here we determined PRCs of putative DA neurons in the substantia nigra pars compacta in brain slices from male and female mice using gramicidin-perforated current-clamp recordings with electrical noise stimuli applied through the patch pipette. On average, and compared with nearby putative GABA neurons, DA neurons showed a low, nearly constant level of sensitivity across most of the ISI, but individual cells had PRCs showing relatively greater sensitivity at early or late phases. Pharmacological experiments showed that DA neuron PRCs are shaped by small-conductance calcium-activated potassium and Kv4 channels, which limit input sensitivity across early and late phases of the ISI. Our results establish the PRC as a tractable experimental measurement of individual DA neuron input-output relationships and identify two of the major ionic conductances that limit perturbations to rhythmic firing. These findings have applications in modeling and for identifying biophysical changes in response to disease or environmental manipulations.

Endoplasmic Reticulum Calcium MediatesDrosophilaWing Development

Background: The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning. However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and bone morphogenetic protein (BMP/Dpp) release for Drosophila wing development. Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology. Ion channels impact development of several tissues and organisms in which BMP signaling is essential. In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin. Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum (ER) Ca++ release into the cytoplasm to regulate the release of BMP. Materials and Methods: To test this hypothesis, we reduced expression of four proteins that control ER calcium, Stromal interaction molecule 1 (Stim), Calcium release-activated calcium channel protein 1 (Orai), SarcoEndoplasmic Reticulum Calcium ATPase (SERCA), small conductance calcium-activated potassium channel (SK), and Bestrophin 2 (Best2) using RNAi and documented wing phenotypes. We use live imaging to study calcium and Dpp release within pupal wings and larval wing discs. Additionally, we employed immunohistochemistry to characterize Small Mothers Against Decapentaplegic (SMAD) phosphorylation downstream of the BMP/Dpp pathway following RNAi knockdown. Results: We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced BMP/Dpp release. Conclusion: Our results suggest control of ER calcium homeostasis is required for BMP/Dpp release, and Drosophila wing development.

Steady-state and transient effects of SK channel block and adrenergic stimulation to counteract acetylcholine-induced arrhythmogenic effects in the human atria: A computational study

Hyperactivity of the parasympathetic nervous system has been linked to the development of paroxysmal atrial fibrillation (AF). The parasympathetic neurotransmitter acetylcholine (ACh) causes a reduction in action potential (AP) duration (APD) and an increase in resting membrane potential (RMP), both of which contribute to enhance the risk for reentry. Research suggests that small-conductance calcium activated potassium (SK) channels may be an effective target for treating AF. Therapies targeting the autonomic nervous system, either alone or in combination with other drugs, have been explored and have been shown to decrease the incidence of atrial arrhythmias. This study uses computational modeling and simulation to examine the impact of SK channel block (SKb) and β-adrenergic stimulation through Isoproterenol (Iso) on countering the negative effects of cholinergic activity in human atrial cell and 2D tissue models. The steady-state effects of Iso and/or SKb on AP shape, APD at 90% repolarization (APD90) and RMP were evaluated. The ability to terminate stable rotational activity in cholinergically-stimulated 2D tissue models of AF was also investigated. A range of SKb and Iso application kinetics, which reflect varying drug binding rates, were taken into consideration. The results showed that SKb alone prolonged APD90 and was able to stop sustained rotors in the presence of ACh concentrations up to 0.01 μM. Iso terminated rotors under all tested ACh concentrations, but resulted in highly-variable steady-state outcomes depending on baseline AP morphology. Importantly, the combination of SKb and Iso resulted in greater APD90 prolongation and showed promising anti-arrhythmic potential by stopping stable rotors and preventing re-inducibility.

Asprosin promotes feeding through SK channel-dependent activation of AgRP neurons.

Asprosin, a recently identified adipokine, activates agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARH) via binding to protein tyrosine phosphatase receptor δ (Ptprd) to increase food intake. However, the intracellular mechanisms responsible for asprosin/Ptprd-mediated activation of AgRPARH neurons remain unknown. Here, we demonstrate that the small-conductance calcium-activated potassium (SK) channel is required for the stimulatory effects of asprosin/Ptprd on AgRPARH neurons. Specifically, we found that deficiency or elevation of circulating asprosin increased or decreased the SK current in AgRPARH neurons, respectively. AgRPARH-specific deletion of SK3 (an SK channel subtype highly expressed in AgRPARH neurons) blocked asprosin-induced AgRPARH activation and overeating. Furthermore, pharmacological blockade, genetic knockdown, or knockout of Ptprd abolished asprosin's effects on the SK current and AgRPARH neuronal activity. Therefore, our results demonstrated an essential asprosin-Ptprd-SK3 mechanism in asprosin-induced AgRPARH activation and hyperphagia, which is a potential therapeutic target for the treatment of obesity.

Bifurcation analysis of motoneuronal excitability mechanisms under normal and ALS conditions.

Bifurcation analysis allows the examination of steady-state, non-linear dynamics of neurons and their effects on cell firing, yet its usage in neuroscience is limited to single-compartment models of highly reduced states. This is primarily due to the difficulty in developing high-fidelity neuronal models with 3D anatomy and multiple ion channels in XPPAUT, the primary bifurcation analysis software in neuroscience. To facilitate bifurcation analysis of high-fidelity neuronal models under normal and disease conditions, we developed a multi-compartment model of a spinal motoneuron (MN) in XPPAUT and verified its firing accuracy against its original experimental data and against an anatomically detailed cell model that incorporates known MN non-linear firing mechanisms. We used the new model in XPPAUT to study the effects of somatic and dendritic ion channels on the MN bifurcation diagram under normal conditions and after amyotrophic lateral sclerosis (ALS) cellular changes. Our results show that somatic small-conductance Ca2+-activated K (SK) channels and dendritic L-type Ca2+ channels have the strongest effects on the bifurcation diagram of MNs under normal conditions. Specifically, somatic SK channels extend the limit cycles and generate a subcritical Hopf bifurcation node in the V-I bifurcation diagram of the MN to replace a supercritical node Hopf node, whereas L-type Ca2+ channels shift the limit cycles to negative currents. In ALS, our results show that dendritic enlargement has opposing effects on MN excitability, has a greater overall impact than somatic enlargement, and dendritic overbranching offsets the dendritic enlargement hyperexcitability effects. Together, the new multi-compartment model developed in XPPAUT facilitates studying neuronal excitability in health and disease using bifurcation analysis.

Restoration of mitochondrial Ca2+ and redox homeostasis by enhancement of SK channels rescues Ca2+cycling in HFpEF.

Heart failure is the leading cause of death in the postindustrial world. While many effective therapies are available to treat heart failure with reduced ejection fraction (HFrEF), these approaches fail to improve outcomes in heart failure with preserved ejection fraction (HFpEF). Small conductance Ca2+-activated K+ (SK) channels have recently emerged as an attractive target to improve defective mitochondrial function and reduce emission of damaging reactive oxygen species (ROS) by the organelle. The goal of the present study was to test enhancement of SK as a new strategy to restore abnormal intracellular Ca2+ cycling and mitochondrial redox and Ca2+ homeostasis in ventricular myocytes from HFpEF rat hearts. To achieve this goal as a model of HFpEF we employed ZSF1 obese rats that demonstrate diastolic dysfunction, unchanged fractional shortening and ejection fraction. The confocal Ca2+ and ROS imaging experiments were carried out in ventricular myocytes isolated from lean and obese ZSF1 rats. Mitochondrial matrix [Ca2+] and ROS were assessed in ventricular myocytes expressing matrix-targeted biosensors mtRCamp1h and MLS-HyPer-7, respectively. To increase SK channel activity, we employed adenovirus-mediated overexpression of rat SK channel type 2 and pharmacological SK channel enhancer NS309 . Periodically paced ventricular myocytes isolated from obese ZSF1 rat hearts exhibited enhanced propensity to spontaneous sarcoplasmic reticulum (SR) Ca2+ release, increased mito-ROS production and a dramatic increase in mitochondrial [Ca2+] when exposed to beta-adrenergic agonist isoproterenol. Enhancement of SK channels in ZSF1 obese myocytes prevented mitochondrial Ca2+ overload, reduced mito-ROS emission to the control levels, and improved cytosolic Ca2+ cycling, reducing diastolic SR Ca2+ release. In summary, enhancement of SK channels shows high therapeutic potential in HFpEF by limiting mitochondrial Ca2+ uptake, thereby reducing oxidative stress restraining excessive SR Ca2+ release during diastole.

Reduction of SK current in rat CPVT model with heterozygous calmodulin 3 mutation D132E is caused by impaired intracellular Ca2+ homeostasis.

Small conductance Ca2+-activated K+ (SK) channels are voltage independent and their gating is governed by Ca2+ via calmodulin (CaM) constitutively bound to the C-terminal CaM-binding domain (CaMBD). Recently, several CaM mutations have been linked to a hereditary arrhythmia syndrome, Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Although in vitro studies demonstrated that CaM mutations cause loss-of-SK-channel complex function, the exact mechanisms remain elusive. The main objective of this work is to delineate potential roles and mechanisms of SK channels dysregulation in CPVT-linked calmodulinopathy. To achieve this goal we generated a novel rat model with CPVT-associated calmodulin 3 (Cam3) point mutation D132E. In vivo ECG recordings of heterozygous CPVT CaM3D132E rats demonstrated increased incidence of epinephrine+caffeine induced ventricular tachycardia. Using whole-cell patch-clamp and confocal Ca2+-imaging we recorded ISK in isolated littermate control (LMC) and D132E myocytes challenged with β-adrenergic agonist isoproterenol under preserved intracellular Ca2+ cycling. Patch-clamp recordings showed reduced ISK in CaMD132E myocytes vs LMCs. However, the amplitude of Ca2+ transients that evoke ISK was also significantly lower in mutant myocytes. To test directly whether CaM3D132E causes SK channels dysfunction we expressed rat SK2 tethered with CaMD132E or WT CaM in rat myocytes. Simultaneous ISK/Ca2+ recordings revealed that this mutation causes two-fold reduction in Ca2+-dependent activation of the channels. In vitro stopped-flow experiments confirmed that CaMD132E mutation impairs CaM interaction with SK2 CaMBD. RNA fluorescence imaging experiments demonstrated lack of CaM3 and SK2/3 mRNAs spatial overlap eliminating possibility of co-translation. These data suggest that CaM3 D132E mutation causes ISK downregulation primarily due to Ca2+ transients amplitude reduction, while direct loss-of-function effects on plasmalemmal SK channels play minimal role given reduced ability of mutant CaM to bind to the channel.

Methods for stable multimeric molecular dynamics simulation and modeling of cardiac SK2 channel function.

Small conductance Ca2+-activated K+ (SK) channels are unique since they are gated solely by changes in intracellular Ca2+ and are known to be upregulated in heart failure. SK channels are regulated via the ubiquitous Ca2+ sensing protein, calmodulin (CaM). Currently there are no structures for SK1-3 channels bound to CaM, thus, significant need for a method to generate stable SK channel models to assess conduction and membrane interactions exists. Recent cryo-EM structures with nearly complete tetrameric SK4-CaM complex (PDB: 6CNM, 6CNN, 6CNO) allows the generation of nearly full-length SK2-CaM homology models (SK2-CaM). Molecular dynamic simulations (MD) performed on SK2-CaM models generated with established methods, however, critical loops and subunits were unstable due to multimeric and size of complex. We have developed a method for each state of SK4-CaM to be used as an input with output being 1μs+ stable MD simulation ofSK2-CaM complex. This process includes many novel approaches using Rosetta, NAMD, and Amber along with specific regional restraints during specific steps; the procedure can differ for various states. MD designed to assess stability and conduction of the relevant SK4-CaM PDBs listed above and comparing results to the similar MD simulations of the final SK2-CaM models, we propose potential mechanisms for ion conduction in SK2 and SK4. In addition, basic residue clustering was detected near the lower leaflet headgroup of the membrane, implying anionic lipid binding locations. SK2-CaM was also stable in all 3 tested states with POPC membrane and varying concentrations of anionic lipids, allowing for the exploration of binding/interacting mechanisms. Using our expertise on the SK2-CaM complex as a template, we propose a new protocol that can be used to create stable MD simulations of homology models for SK1-3 using SK4 as an input.

Sex-specific contribution of small-conductance Ca2+-activated K+ current to atrial electrophysiology and arrhythmogenesis.

Small-conductance Ca2+-activated K+ (SK) channels are predominantly expressed in the atria, thus offering a unique therapeutic target for atrial fibrillation (AF) independent of ventricular function. Several studies have reported remodeling of SK channels in AF, whereby SK current is increased during AF-induced remodeling in several animal models. Nevertheless, data on SK current (ISK) in atrial myocytes from patients remain scarce, and the AF-associated changes are controversial. Both upregulation and downregulation have been reported in chronic AF vs. normal sinus rhythm, accompanied by increased or decreased action potential duration (APD) lengthening by SK-channel inhibition. A potential confounding factor is the observation that sex-specific differences exist in ISK function in the ventricles, which could also be present in the atria, though this remains unknown.

Physiological role of SK channels in modulating cardiac repolarization: APD and dispersion at slow heart rate in long QT syndrome.

Long QT syndrome (LQTS) is a congenital disease associated with polymorphic ventricular tachycardia (pVT) and sudden cardiac death. The hallmarks of LQTS are early afterdepolarizations that initiate pVT and dispersion of repolarization allowing reentry formation. Small conductance Ca2+-activated K+ (SK) channels are directly gated by Ca2+ and provide feedback from Ca2+ to membrane voltage to accelerate cardiac repolarization, which may help normalizing LQTS. Using optical mapping, we examined the role of ISK in transgenic rabbit models of LQT1, LQT2, and LQT5 and found that enhancing ISK with NS309 consistently shortened APD, reduced APD dispersion, and suppressed pVT genesis universally in the three LQT rabbit models. Enhancement of ISK has a greater impact at longer cycle length, flattening restitution curves at slow heart rate where most pVTs occur in LQTS. We incorporated a six state ISK gating simulation combined with intrinsic rectification and divalent block into the Mahajan rabbit action potential model to investigate the role of ISK in suppressing LQT-related arrhythmias. Increasing levels of SK channel conductance (gSK) were able to shorten and reduce APD dispersion simulated by conductance gradients of gKs by ∼64% under LQT2 conditions and gtof by ∼70% in LQT1 conditions. Computer modeling indicates that ISK can reduce APD dispersion through two mechanisms; increased Ca2+ duration in the prolonged APD cells augment ISK to shorten long APD and lowering plateau Vm by ISK masks IKs heterogeneity by reducing IKs's role in repolarization. In conclusion, the Ca2+-dependent gating of ISK allows it to directly feedback on the APD especially when other K+ channels are not functional and to be a potential therapeutic target for LQTS.

Histamine bidirectionally regulates the intrinsic excitability of parvalbumin-positive neurons in the lateral globus pallidus and promotes motor behaviour.

Parvalbumin (PV)-positive neurons are a type of neuron in the lateral globus pallidus (LGP) which plays an important role in motor control. The present study investigated the effect of histamine on LGPPV neurons and motor behaviour. Histamine levels in LGP as well as its histaminergic innervation were determined through brain stimulation, microdialysis, anterograde tracing and immunostaining. Mechanisms of histamine action were detected by immunostaining, single-cell qPCR, whole-cell patch-clamp recording, optogenetic stimulation and CRISPR/Cas9 gene-editing techniques. The effect of histamine on motor behaviour was detected by animal behavioural tests. A direct histaminergic innervation in LGP from the tuberomammillary nucleus (TMN) and a histamine-induced increase in the intrinsic excitability of LGPPV neurons were determined by pharmacological blockade or by genetic knockout of the histamine H1 receptor (H1 R)-coupled TWIK-related potassium channel-1 (TREK-1) and the small-conductance calcium-activated potassium channel (SK3), as well as by activation or overexpression of the histamine H2 receptor (H2 R)-coupled hyperpolarization-activated cyclic nucleotide-gated channel (HCN2). Histamine negatively regulated the STN → LGPGlu transmission in LGPPV neurons via the histamine H3 receptor (H3 R), whereas blockage or knockout of H3 R increased the intrinsic excitability of LGPPV neurons. Our results indicated that the endogenous histaminergic innervation in the LGP can bidirectionally promote motor control by increasing the intrinsic excitability of LGPPV neurons through postsynaptic H1 R and H2 R, albeit its action was negatively regulated by the presynaptic H3 R, thereby suggesting possible role of histamine in motor deficits manifested in Parkinson's disease (PD).

Downregulation of ICCs and PDGFRα+ cells on colonic dysmotility in hirschsprung disease.

To investigate the effect of the distribution and expression of interstitial cells of Cajal (ICCs) and platelet-derived growth factor receptor-α positive (PDGFRα+) cells in different colon segments on colonic motility in children with Hirschsprung disease (HSCR). Smooth muscles of the narrow and dilated segments of the colon were obtained from 16 pediatric patients with HSCR. The proximal margin was set as the control section. The mRNA and protein expressions of c-Kit, PDGFRα, ANO1, and SK3 channels were examined. Circular smooth muscle strips of the colon were prepared for performing electrophysiology experiments using electric field stimulation (EFS) and intervention from different drugs (TTX, NPPB, Apamin, L-NAME, and CyPPA). The mRNA and protein expressions of c-Kit, ANO1, PDGFRα, and SK3 were much lower in the narrow segment than those in the dilated and proximal segments of the colon. The narrow segment showed a considerably spontaneous contraction of the muscle strip. After the EFS, the relaxation response decreased from the proximal to the narrow segment, whereas the contraction response increased. TTX blocking did not cause any significant changes in the narrow segment. In contrast, when NPPB, Apamin, L-NAME, and CyPPA were used to intervene in the muscle strips, the proximal segment showed a more sensitive inhibitory or excitatory response than the narrow segment. Downregulation of the ICCs and PDGFRα+ cells from the proximal to narrow segment may be responsible for the dysmotility of the colon in pediatric HSCR.

Endothelial IK and SK channel activation decreases pulmonary arterial pressure and vascular remodeling in pulmonary hypertension.

Endothelial cells (ECs) from small pulmonary arteries (PAs) release nitric oxide (NO) and prostacyclin, which lower pulmonary arterial pressure (PAP). In pulmonary hypertension (PH), the levels of endothelium-derived NO and prostacyclin are reduced, contributing to elevated PAP. Small-and intermediate-conductance Ca2+-activated K+ channels (IK and SK)-additional crucial endothelial mediators of vasodilation-are also present in small PAs, but their function has not been investigated in PH. We hypothesized that endothelial IK and SK channels can be targeted to lower PAP in PH. Whole-cell patch-clamp experiments showed functional IK and SK channels in ECs, but not smooth muscle cells, from small PAs. Using a SU5416 plus chronic hypoxia (Su + CH) mouse model of PH, we found that currents through EC IK and SK channels were unchanged compared with those from normal mice. Moreover, IK/SK channel-mediated dilation of small PAs was preserved in Su + CH mice. Consistent with previous reports, endothelial NO levels and NO-mediated dilation were reduced in small PAs from Su + CH mice. Notably, acute treatment with IK/SK channel activators decreased PAP in Su + CH mice but not in normal mice. Further, chronic activation of IK/SK channels decreased PA remodeling and right ventricular hypertrophy, which are pathological hallmarks of PH, in Su + CH mice. Collectively, our data provide the first evidence that, unlike endothelial NO release, IK/SK channel activity is not altered in PH. Our results also demonstrate proof of principle that IK/SK channel activation can be used as a strategy for lowering PAP in PH.

Calcium dependence of both lobes of calmodulin is involved in binding to a cytoplasmic domain of SK channels.

KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+. A model for SK gating proposes that the N-terminal domain (N-lobe) of calmodulin is required for activation, but an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Although structures support a domain-driven hypothesis of SK gate activation by calmodulin, only a partial understanding is possible without measuring both channel activity and protein binding. We measured SK2 (KCa2.2) activity using inside-out patch recordings. Currents from calmodulin-disrupted SK2 channels can be restored with exogenously applied calmodulin. We find that SK2 activity only approaches full activation with full-length calmodulin with both an N- and a C-lobe. We measured calmodulin binding to a C-terminal SK peptide (SKp) using both composition-gradient multi-angle light-scattering and tryptophan emission spectra. Isolated lobes bind to SKp with high affinity, but isolated lobes do not rescue SK2 activity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe-binding affinity is strong independent of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to both the N- and C-lobes of calmodulin at Ca2+ concentrations that activate SK2, demonstrating that the C-lobe interaction with SKp changes with Ca2+. Our peptide-binding data and electrophysiology show that SK gating models need deeper scrutiny. We suggest that the Ca2+-dependent associations of both lobes of calmodulin to SKp are crucial events during gating. Additional investigations are necessary to complete a mechanistic gating model consistent with binding, physiology, and structure.

Molecular dynamics simulations of the calmodulin-induced α-helix in the SK2 calcium-gated potassium ion channel

The family of small-conductance Ca2+-activated potassium ion channels (SK channels) is composed of four members (SK1, SK2, SK3, and SK4) involved in neuron-firing regulation. The gating of these channels depends on the intracellular Ca2+ concentration, and their sensitivity to this ion is provided by calmodulin (CaM). This protein binds to a specific region in SK channels known as the calmodulin-binding domain (CaMBD), an event which is essential for their gating. While CaMBDs are typically disordered in the absence of CaM, the SK2 channel subtype displays a small prefolded α-helical region in its CaMBD even if CaM is not present. This small helix is known to turn into a full α-helix upon CaM binding, although the molecular-level details for this conversion are not fully understood yet. In this work, we offer new insights on this physiologically relevant process by means of enhanced sampling, atomistic Hamiltonian replica exchange molecular dynamics simulations, providing a more detailed understanding of CaM binding to this target. Our results show that CaM is necessary for inducing a full α-helix along the SK2 CaMBD through hydrophobic interactions with V426 and L427. However, it is also necessary that W431 does not compete for these interactions; the role of the small prefolded α-helix in the SK2 CaMBD would be to stabilize W431 so that this is the case. In conclusion, our findings provide further insight into a key interaction between CaM and SK channels that is important for channel sensitivity to Ca2+.

CRAC and SK Channels: Their Molecular Mechanisms Associated with Cancer Cell Development.

Cancer represents a major health burden worldwide. Several molecular targets have been discovered alongside treatments with positive clinical outcomes. However, the reoccurrence of cancer due to therapy resistance remains the primary cause of mortality. Endeavors in pinpointing new markers as molecular targets in cancer therapy are highly desired. The significance of the co-regulation of Ca2+-permeating and Ca2+-regulated ion channels in cancer cell development, proliferation, and migration make them promising molecular targets in cancer therapy. In particular, the co-regulation of the Orai1 and SK3 channels has been well-studied in breast and colon cancer cells, where it finally leads to an invasion-metastasis cascade. Nevertheless, many questions remain unanswered, such as which key molecular components determine and regulate their interplay. To provide a solid foundation for a better understanding of this ion channel co-regulation in cancer, we first shed light on the physiological role of Ca2+ and how this ion is linked to carcinogenesis. Then, we highlight the structure/function relationship of Orai1 and SK3, both individually and in concert, their role in the development of different types of cancer, and aspects that are not yet known in this context.

Venom composition and pain-causing toxins of the Australian great carpenter bee Xylocopa aruana

Most species of bee are capable of delivering a defensive sting which is often painful. A solitary lifestyle is the ancestral state of bees and most extant species are solitary, but information on bee venoms comes predominantly from studies on eusocial species. In this study we investigated the venom composition of the Australian great carpenter bee, Xylocopa aruana Ritsema, 1876. We show that the venom is relatively simple, composed mainly of one small amphipathic peptide (XYTX1-Xa1a), with lesser amounts of an apamin homologue (XYTX2-Xa2a) and a venom phospholipase-A2 (PLA2). XYTX1-Xa1a is homologous to, and shares a similar mode-of-action to melittin and the bombilitins, the major components of the venoms of the eusocial Apis mellifera (Western honeybee) and Bombus spp. (bumblebee), respectively. XYTX1-Xa1a and melittin directly activate mammalian sensory neurons and cause spontaneous pain behaviours in vivo, effects which are potentiated in the presence of venom PLA2. The apamin-like peptide XYTX2-Xa2a was a relatively weak blocker of small conductance calcium-activated potassium (KCa) channels and, like A. mellifera apamin and mast cell-degranulating peptide, did not contribute to pain behaviours in mice. While the composition and mode-of-action of the venom of X. aruana are similar to that of A. mellifera, the greater potency, on mammalian sensory neurons, of the major pain-causing component in A. mellifera venom may represent an adaptation to the distinct defensive pressures on eusocial Apidae.

Activation of small conductance Ca2+ -activated K+ channels suppresses Ca2+ transient and action potential alternans in ventricular myocytes.

At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We tested the hypothesis that in single rabbit ventricular myocytes pharmacological modulation of SK channels plays a causative role for the development of pacing-induced CaT and AP duration (APD) alternans. SK channel blockers (apamin, UCL1684) had only a minor effect on AP repolarization. However, SK channel activation by NS309 resulted in significant APD shortening, demonstrating that functional SK channels are well expressed in ventricular myocytes. The effects of NS309 were prevented or reversed by apamin and UCL1684, indicating that NS309 acted on SK channels. SK channel activation abolished or reduced the degree of pacing-induced CaT and APD alternans. Inhibition of KV 7.1 (with HMR1556) and KV 11.1 (with E4031) channels was used to mimic conditions of long QT syndromes type-1 and type-2, respectively. Both HMR1556 and E4031 enhanced CaT alternans that was prevented by SK channel activation. In AP voltage-clamped cells the SK channel activator had no effect on CaT alternans, confirming that suppression of CaT alternans was caused by APD shortening. APD shortening contributed to protection from alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest that SK activation could be a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy for patients with long QT syndrome. KEY POINTS: At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and intracellular Ca2+ release amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We investigated whether pharmacological modulation of SK channels affects the development of cardiac alternans in normal ventricular cells and in cells with drug-induced long QT syndrome (LQTS). While SK channel blockers have only a minor effect on AP morphology, their activation leads to AP shortening and abolishes or reduces the degree of pacing-induced Ca2+ and AP alternans. AP shortening contributed to protection against alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest SK activation as a potential intervention to avert the development of alternans with important ramifications for arrhythmia prevention for patients with LQTS.

Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)] produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.